The major goal of this proposal is to investigate the mechanism of action of conjugated linoleic acids (CLA), the naturally occurring isomers of linoleic acid (LA), which have several unique physicochemical properties and biological activities. The two major CLA isomers in the dairy products and meats, namely cis 9 trans 11 CLA, and trans 10 cis 12 CLA, have several isomer-specific and species-specific beneficial effects, including anti-cancer and anti-obesity effects. However, they are also known to cause undesirable effects such as insulin resistance and fatty liver in certain species but not others. Despite extensive studies, the mechanism of action of CLA is largely unknown. In this proposal, we will test the novel hypothesis that the CLA act through modification of the membrane structure, especially the raft domains of the plasma membrane, and the micro environment of the endoplasmic reticulum (ER). We will first determine the incorporation profiles of different CLA isomers into membranes of cultured hepatocytes, adipocytes, and myocytes of human and mouse origins. These studies will determine to what extent specific CLA isomers modify the lipid and fatty acid composition of the raft domains and the ER, and whether there is a tissue- and species- specificity in the incorporation profiles and membrane composition changes. We will also investigate the effect of the presence of CLA at the sn-1 position of phospholipids on the properties of the membranes, since our preliminary studies show that unlike LA, the CLA are preferentially incorporated into the sn-1 position of cellular phospholipids. We will next compare the in vivo incorporation profiles of dietary CLA in mice and rats, since these two animal species respond very differently to CLA, especially with regard to insulin resistance and fatty liver development. The effect of CLA on the distribution of raft-associated proteins will be investigated in cultured cells, as well as in primary adipocytes and hepatocytes from mice and rats. The function of raft associated proteins, specifically those involved in the insulin receptor signaling pathway will be studied following the CLA enrichment and insulin stimulation of the cells in vitro. In addition, the effectof CLA enrichment of cell membranes on glucose uptake, glycogenesis, and lipogenesis will be assessed. These studies should show whether the isomer- specific effects of CLA on insulin resistance are due to their modification of membrane structure. Finally, we will test the hypothesis that specific CLA species induce ER stress, and consequently promote the development of fatty liver and insulin resistance. We propose that the previously reported effects of CLA on the transcription of various genes involved in lipid metabolism and inflammation are due to the altered membrane composition of ER, which in turn affects the activation of transcription factors including sterol regulatory element binding proteins (SREBPs), the master regulators of lipid metabolism. The effect of various CLA isomers on ER lipid composition and the induction of ER stress proteins will be correlated with the activation of the SREBPs and the expression of their target genes. The results from all these studies should lead to a better understanding of the isomer-specific, tissue-specific, and species-specific actions of CLA, and to a more rational use of these nutraceutical agents for treatment or prevention of obesity, diabetes and other diseases.